The PI's laboratory has recently uncovered the existence of a hitherto unknown family of genes present in organisms as diverse as mice, flies, and nematodes with a shared domain of homology also present in the classical mouse T locus. This domain has been named the T-box. The T-box region of the T locus product has been shown by other workers to have sequence- specific DNA binding activity. A role for the T locus product in regulating the proper development of a portion of the mesodermal lineage has been elucidated from results obtained by other investigators with mice, frogs, and zebrafish. In studies of the first three newly uncovered mouse members of the T-box family, unique patterns of temporal and spatial expression were demonstrated in the developing embryo. The working hypothesis is that the new T-box genes, like the prototypic al T locus, play critical roles in developmental signaling. The primary experimental objectives of this proposal and the companion application from Dr. Papaioannou are to identify the complete set of mouse T-box genes, investigate their evolutionary history, and determine the role that each plays in development. However, the overall goal is broader in scope and is aimed at a holistic view of the complete gene system, with an understanding of the structural and functional relationships that exist among the various T-box genes and, perhaps, the fundamental role that the T-box motif may have played in the evolution of developmental complexity. To accomplish this task will require investigations along the lines of genetics, molecular biology, evolution, and experimental embryology to be performed at Princeton and Columbia. The specific aims of this proposal are; (1) Cloning nd characterization of the complete mouse T-box gene gamily. (2) Genetic analysis of mouse T-box genes to allow the identification and pursuit of candidate mouse mutations or human diseases caused by variant T- box alleles and the identification of gene clusters for further characterization through genomic analysis. T-box interactions with other genes will also be pursued through the mapping of modifier loci. (3) Understanding the evolution of the T-box gene family by cloning T-box homologs from a series of model organisms at different points in the metazoan phylogenetic tree. (40 Elucidating the functions of the T-box gene products through the identification of DNA binding sites and, ultimately, downstream target genes.